Annals of Agricultural Science (2015) 60(1), 95–104

H O S T E D BY
Faculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

Biotechnological applications of fungal endophytes

associated with medicinal plant Asclepias sinaica
(Bioss.)
Amr Hamza Fouda 1, Saad El-Din Hassan *, Ahmed Mohamed Eid 2,
Emad El-Din Ewais 3

Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt

Received 19 March 2015; accepted 5 April 2015

Available online 12 May 2015

KEYWORDS Abstract Fungal endophytes associated with medicinal plants have potential role to promote plant
Endophytes; growth through different mechanisms. However, the biological and ecological roles of fungal endo-
Indole acetic acid; phytes still totally unexplored. In this study, three different fungal endophytes were isolated from
Extracellular enzyme; the medicinal plant of Asclepias sinaica and identified as Penicillium chrysogenum Pc_25,
Medicinal plants Alternaria alternata Aa_27 and the third fungal strain was described as sterile hyphae Sh_26. It
was recorded that, these endophytes had various ability to produce several extracellular enzymes
including amylase, pectinase, cellulase, gelatinase, xylanase and tyrosinase. Their antimicrobial
activities against different specific test organisms were investigated as well. In addition, both endo-
phyte isolates i.e. Sh_26 and Aa_27 were found to promote root growth higher than Pc_25 and con-
trol treatments. These fungal isolates had a considerable impact on plant growth parameters
including root elongation as a result of ammonia and IAA production.
ª 2015 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams
University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
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Introduction plant tissues for all or part of their life cycle. Endophytes have
the ability to colonize internal plant tissues of healthy leaves,
Endophytes are defined as microorganisms including bacteria, petioles, stems, twigs, bark, root, fruit, flower, and seeds without
fungi, and actinomycetes that inhabit intra- and intercellular causing any apparent harm or pathogenic infection to their host
plants. Endophytic fungi are ecological and polyphyletic group
* Corresponding author. Tel.: +20 1023884804.
of highly diverse fungi, mostly belonging to ascomycetes and
E-mail addresses: amr.fouda83@gmail.com (A.H. Fouda), Saad.
anamorphic fungi (Arnold, 2007). Approximately, it has been
el-din.hassan@umontreal.ca (S.E.D. Hassan), medoahmedwave@
yahoo.com (A.M. Eid), Ewais_e@yahoo.com (E.E.-D. Ewais). estimated that more than one million different endophytic fun-
1
Tel.: +20 1113351244. gal strains inhabit about 300,000 various plant species. The
2
Tel.: +20 1223360234. hyperdiversity of endophytic fungi derives from that each indi-
3
Tel.: +20 1119636529. vidual plant species can be colonized with one or more fungal
Peer review under responsibility of Faculty of Agriculture, Ain-Shams strains (Huang et al., 2007). Fungal endophytes produce
University.
http://dx.doi.org/10.1016/j.aoas.2015.04.001
0570-1783 ª 2015 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
96 A.H. Fouda et al.

bioactive metabolites that mediate in the plant–endophyte inter- Materials and methods
action (Strobel, 2003). In addition, fungal endophytic metabo-
lites are useful resources for natural products which effectively Plant sampling and study area
have wide range of application in medicine, agriculture, and
industry (strobel and Daisy 2003; Selim et al., 2012).
Plants of A. sinaica were collected from Ain Shakaya
Fungal endophytes have the ability to produce numerous
(28.543386 N, and 33.933071 E), Saint Katherine, South
extracellular enzymes; such as pectinases, cellulases, lipases,
Sinai, Egypt. The plant materials were carefully placed in ster-
amylases, laccases, and proteinases. These fungal enzymes play
ile polyethylene bags and brought to the laboratory in portable
the key role in biodegradation and hydrolysis processes which
cool chambers (4 C). The botanical identification of these
are significantly important mechanisms against pathogenic
plants has been carried out at the herbarium of Botany and
infection and to obtain their nutritional need from the host
Microbiology Department of Al-Azhar University, and speci-
plants (Sunitha et al., 2013). The ability of fungal endophytes
men of the plant herbarium is deposited in the herbarium of
to produce different enzymes has been reported by Choi et al.
Botany and Microbiology Department of Al-Azhar
(2005) and Sunitha et al. (2013), however further quantitative
University under the registration name of A. sinaica (1_As).
assays for fungal endophytic enzymes are required to under-
Plant picture is shown in Plate 1.
stand the ecological role of these fungal endophytes.
Many bioactive metabolites are originated from microbial
Isolation of fungal endophytes
organisms, fungi are the core important groups of eukaryotic
organisms that have wide capacity to produce numerous
metabolites with antimicrobial activities and possess potential The plant leaves were washed by running tap water and steril-
application as drugs. Several bioactive compounds including ization of leaves surfaces was achieved by subsequent soaking
antifungal and antibacterial agents have been isolated from them in series of solutions as follows: sterile distilled water for
fungi (Suryanarayanan et al., 2009). However few endophytic 1 min, ethanol 70% for 1 min, sodium hypochlorite 2.5% for
fungal isolates have investigated for their biological applica- 4 min, ethanol 70% for 30 s and finally washed in sterile dis-
tions including their ability for antimicrobial activity; thus, it tilled water for 3 times. The last washing water was plated onto
seems that screening the antimicrobial activity of fungal endo- bacterial, fungal, and actinomycetales culture media of
phytes is valuable to discover novel antimicrobial producers. Nutrient agar, Czapek Dox agar, and Starch nitrate agar,
Promotion of plant growth is the major contribution of respectively. The success of surface sterilization method was
fungal symbiosis (Hassan et al., 2013), however fungal endo- confirmed by the absence of any microbial growth onto the
phytes promote plant growth through the production of cultural media from the plating of last washing water.
ammonia and phytohormones, particularly indole acetic acid The sterilized plant leaves were cut by sterilize knife into
(IAA) (Bal et al., 2013). Generally, IAA acts as plant growth 5 mm segments. Twenty leaf segments were placed in Petri
promoter which enhances both cell elongation and cell divi- dishes (9 cm) containing Czapek Dox agar and Malt extract
sion, and is essential for plant tissues differentiation agar media and incubated at 28 ± 2 C. Another part of ster-
(Taghavi et al., 2009). The ability of soil microorganisms to ilized leaves segments were crushed in sterile saline solution by
involve in the synthesis of IAA in pure culture and in soil sterile homogenizer. One ml of sterilized crushed samples was
has been recorded (Spaepen and Vanderleyden, 2011); how- serially diluted till 10 3 and 0.1 ml was spread onto Czapek
ever, endophytic microorganisms isolated from various plants Dox agar and Malt extract agar media and incubated at
have showed high IAA production level compared to those 28 ± 2 C (Arora et al., 2014). Regular observations were
isolated from root-free soil (Spaepen et al., 2007). The func- done from the second day onwards for a period of 3–4 weeks
tional role of IAA in plant growth in addition to the capacity for fungal growth (Bills and Polishook, 1991). The fungal
of fungal endophytes to produce IAA has gained great atten-
tion due to their impact on the concentration and distribution
of IAA in plant tissues.
Little is known about the biology and ecology of fungal
endophytes; subsequently, isolation and characterization of
fungal endophytes that colonize different plant species of
various habitats and ecosystem is potentially useful.
Asclepias sinaica is a medicinal plant from the Sinai desert is
widely used in traditional Bedouin medicine to treat some can-
cer diseases (El-Seedi et al., 2013). A. sinaica plant may host
useful microbial community that might be potentially have
several biological and ecological role in their environment.
However, information about the biological and ecological role
of fungal endophytes community of A. sinaica plant is still
unknown. Therefore, the aim of this study was to isolate and
identify fungal endophytes survived inside the leaves of A.
sinaica plant, to determine the antimicrobial and extracellular
enzymatic activities of these fungal endophytes, to assay the
capacity of these endophytes to produce ammonia and IAA,
and finally to estimate the effect of fungal inoculation on the
growth of plant roots. Plate 1 Asclepias sinaica (Bioss.).
Biotechnological applications of fungal endophytes
growth from internal tissues or crushed segments was checked
for purity, transferred to fresh cultural slants and stored at
4 C for further study.
Identification of fungal endophytes
Identification was done on the basis of morphological, taxo-
nomic characteristics and direct microscopic examination of
the fruiting bodies and spores using standard manuals.
(Moubasher and Moustafa, 1970; Pitt and Hocking, 1997 for
penicillium sp., Domsch et al., 1980 for sterile hyphae;
Woudenberg et al., 2013, for alternarea sp.).
Antimicrobial activity of fungal endophytes
Preparation of endophytes extracts
To test the antimicrobial activity of fungal endophytes, the
isolated fungal strains were cultured in Malt extract liquid
medium for 10 days at 28 C on a shaker at 180 rpm. Crude
fermentation broth was blended thoroughly and centrifuged
at 4000 rpm for 5 min. Liquid supernatant was extracted with
an equal volume of ethyl acetate thrice. The organic solvent
extract was then evaporated under reduced pressure using
rotary evaporator. The crude extracts were dissolved in
dimethyl sulfoxide (DMSO) and used for antimicrobial screen-
ing (Lv et al., 2010).
Antimicrobial assay of fungal endophytes extracts
The crude extracts of fungal endophytes were dissolved in
DMSO and used for antimicrobial activity assay.
Antimicrobial activity of secondary crude metabolites was
tested by well diffusion method according to the protocol of
Yadav et al. (2010).
The coded test organisms used for antimicrobial assay were
Gram-positive bacteria: Staphylococcus aureus, ATCC 6538
and Bacillus subtilis, ATCC 6633; Gram-negative bacteria:
Escherichia coli, ATCC 8739; Pseudomonas aeruginosa,
ATCC 9027 and Salmonella typhimurium ATCC 14028; and
yeast strain of Candida albicans, ATCC 10231. Test organisms
inoculated in Petri dishes containing Muller-Hinton agar med-
ium for bacteria, and sabouraud agar medium for yeast.
Three wells of 1 cm diameter were made in the test organ-
ism growing media using a sterile cork borer and filled with
40 ll of fungal endophytic extract. Negative control wells were
filled with 40 ll of DMSO without endophytic fungal extracts.
The plates were kept in refrigerator at 4 C for 4 h for complete
diffusion of antimicrobial compounds, and then incubated at
35 ± 2 C for 24 h for bacteria and 28 ± 2 C for yeast. The
presence of inhibition zones around the wells was measured
to determine the antimicrobial activity of fungal endophytic
secondary extracts. All antimicrobial activity assays were per-
formed in triplicate.
Screening the extracellular enzymatic activities of fungal
endophytes
The production and activity of extracellular enzymes by fungal
endophytes were assessed by growing them on Yeast-Malt
(YM) agar media (YM: 10 g/L glucose, 5 g/L peptone, 3 g/L
yeast extract, 3 g/L malt extract, 1.5% agar, pH 6.7) (Molina
97
et al., 2012) and placing 5 mm fungal plugs on the YM agar
media supplemented with dissolved and specific indicative sub-
strates. After incubation for 3–5 days depending on the growth
rates of fungal endophytes at 28 C, the appearance of clear
zone surrounding the fungal colony was measured after adding
specific reagent and used as indicator for extracellular enzy-
matic activities. All assays were performed in triplicate.
Amylolytic activity
Amylase activity was assessed by growing the fungal isolates
on YM agar medium supplemented with 1% soluble starch.
After incubation, the plates were flooded with 1% iodine.
The appearance of clear zones around fungal growth was mea-
sured to determine the amylolytic activity.
Proteolytic activity
The YM agar medium containing 1% gelatine was used to
determine the fungal protease enzyme activity. After incuba-
tion, the degradation of gelatine was seen as clear zoon around
the colonies by using acidic mercuric chloride as an indicator.
Cellulase activity
The appearance of clear zone around the fungal colony grown
on YM medium supplemented with 1% cellulose or carboxy-
methylcellulose (CMC) was measured, in order to assess the
fungal cellulolytic activity after adding iodine solution as
indicator.
Pectinolytic activity
Pectinolytic activity was determined by growing the fungi in
1% Pectin-YM containing medium. After the incubation
period, the plates were flooded with 1% aqueous solution of
hexadecyl trimethyl ammonium bromide. A clear zone formed
around the fungal colony indicated the activity of pectinase
enzyme.
Xylanolytic activity
Yeast-Malt agar medium supplemented with 1% xylan of corn
cobs was used in order to measure the fungal xylanolytic activ-
ity. After incubation period, screening the xylanase activity
was appeared as a clear zone around the fungal colony as a
result of using absolute ethyl alcohol to indicate the xylan
biodegradation.
Tyrosinase activity
The tyrosinase activity of endophytic fungal strains was
assessed by growing fungi on YM agar supplemented with
1% tyrosine. After 5 days of incubation period, the mixture
of 0.1% q-cresol and 0.05% glycin was overlaid on the surface
of colony. The appearance of reddish brown colour around the
fungal colony indicated the activity of tyrosinase enzyme
(Hankin and Anagnostakis, 1975).
IAA Production by fungal endophytes
The ability of fungal endophytes to produce IAA was deter-
mined, where the endophytic fungal strains were inoculated
in Czapek Dox (CD) broth at 28 C. One disc (1 cm diameter)
of each inoculum was added to 20 ml of CD liquid medium
98
containing different concentration of tryptophan (1, 2 and
5 mg/ml) or without tryptophan and incubated for 10 days.
Five ml of each culture was collected from the incubating
broth after 10 days and centrifuged at 6000 rpm for 30 min.
One ml of the supernatant was mixed with 1 drop of
orthophosphoric acid and 2 ml of Salkowski’s reagent
(300 ml concentrated Sulphuric acid; 500 ml distilled water;
15 ml 0.5 M FeCl3). Development of a pink colour indicated
IAA production as the optical density was measured at
530 nm using Spectrophotometer (Jenway 6305 UV spec-
trophotometer). The amount of IAA produced was estimated
by a standard IAA graph (Ahmad et al., 2005). All the IAA
production and determination were performed in triplicate.
Ammonia production
The ability of Endophytic fungal strains to produce ammonia
was assessed, where fungal strains were grown in peptone
water for 72 h at 28 C. The addition of 1 ml Nessler’s reagent
in peptone liquid media was used to determine the ammonia
production by endophytes. Where, the colour change to faint
yellow indicates the minimum ammonia production and col-
our change into the deep yellow to brownish colour indicates
the maximum ammonia production by endophytes (Singh
et al., 2014).
Effect of fungal endophytes on root growth
The effect of endophytic inoculation on root growth was inves-
tigated on maize (Zea mays L.) plant. The seeds of maize (Zea
mays L.) were surface sterilized by soaking in 2.5% sodium
hypochlorite for 3 min and then they were washed by sterile
Fig. 1 Cultural morphology and microscopic picture of fungal endophyte Pc_25 grown on CYA and 2% MEA media (X = 800).
A.H. Fouda et al.
distilled water for 5 times. Endophytic fungal strains were
inoculated in CD broth at 28 ± 2 C for 5 days. Sterilized
seeds were incubated with 50 ml of each fungal strain broth
medium at room temperature for 24 h. The CD broth without
fungal inoculation was used as control treatment. After 24 h
incubation, the soaked seeds were placed in sterilized cup con-
taining wet sterilized filter paper and they were incubated at
room temperature for 5 days in dark to measure the root
growth.
Statistical analysis
All results presented are the means of three independent repli-
cates. Data were subjected to statistical analysis by a statistical
package SPSS v17. The mean difference comparison between
the treatments was analysed by analysis of variance
(ANOVA) and subsequently by Tukey HSD test at P < 0.05.
Results
Isolation and identification of fungal endophytes
Three fungal endophytes Pc_25, Sh_26, and Aa_27 were iso-
lated from A. sinaica leaves, these fungal strains were identified
as Penicillium chrysogenum, one fungal strain was described as
Sterile hyphae, and Alternaria alternata, respectively.
Morphological description of Pc_25 (Fig. 1 and Table 1)
showed that the colonies on MEA had 25–40 mm diameter,
usually plane, low and velutinous, occasionally floccose cen-
trally or somewhat granular; mycelium inconspicuous; conidial
production moderate to heavy, greyish turquoise to dull green;
reverse pale, yellowish, yellow brown. Conidiophores are
Biotechnological applications of fungal endophytes 99

borne from surface or subsurface hyphae, stipes commonly to low and dense, dark olive to dark in old culture; reverse
200–300 lm long, with thin smooth walls, penicilli typically brown to nearly black. Conidia are blown out from the apices
terverticillate, with 1–2 rami, either terminal and appressed of undistinguished conidiophores as short, irregularly
or sometimes subterminal and divergent, in that case appear- branched chains of up to 10 units, and then septating both lat-
ing biverticillate; phialides ampulliform, 7–8(–10) lm long; erally and longitudinally, with up to six transverse and two to
conidia ellipsoidal to subspheroidal, 2.5–4.0 lm long, smooth three longitudinal or oblique septa, usually of clavate or pyri-
walled, borne in long, irregular columns. form shape overall, tapering towards the apices, forming a
The morphology of second fungal strain, of Sh_26 (Fig. 2) short beak, in culture usually 20–40 and 8–12 lm, with walls
showed that the colonies appeared on MEA medium reached smooth to conspicuously roughened.
to 45–65 mm diameter, dense to floccose white mycelium,
reverse uniformly pale or white centrally, colourless at the Antimicrobial activity
margins, branched nonseptated mycelium.
Fig. 3 shows that colonies of Aa_27, grown on MEA were The antimicrobial activities of fungal endophytes isolates were
50–70 mm diameter, or covering the whole Petri dish, plane, of evaluated. Analyses of variance (ANOVA) revealed that there
deeply floccose off-white to grey brown mycelium converting were significant differences and variations among the three
fungal endophytes to inhibit the growth of the coded tested
microorganisms (Fig. 4).
Multi-comparison analysis of the differences showed that
Table 1 Morphological characters of fungal endophyte Pc_25 the crude extract of Sh_26 significantly inhibited the growth
identified as Penicillium chrysogenum. of S. aureus, B. subtilis, E. coli, P. aeruginosa, and S.
Typhimurium, compared to control and Pc_25 and Aa_27
Conidia diameter 2.5–4 lm endophyte extracts (F3,8 = 366.75, 3481.0, 16.616, 968.0, and
Conidial ornamentation Smooth
27.848; P 6 0.001) (Fig. 5). However, the Sh_26 did not signif-
Phialide length 7–8 (10) lm
Metulae length 7–8 (10) lm
icantly inhibit the growth of C. albicans compared to control
Stipe width 2–3 lm treatment (F3,8 = 2703.9; P = 1.00).
Stipe ornamentation Smooth Inhibition zone of secondary metabolites from fungal endo-
Conidiophore branching pattern Terverticillate phyte of Aa_27 against P. aeruginosa and S. aureus was signif-
icantly higher (P 6 0.001) than those observed for the control
Macromorphology on MEA
Colony diameter 25–40 mm
and Pc_25 endophytic treatments. Moreover, the only endo-
Colour obverse Dull green phytic fungal extract which inhibited the growth of tested
Colour reverse Pale yellow, yellowish brown microbe of C. albicans was Aa_27 endophyte (Fig. 5).
Results also showed that Endophyte of Aa_27 did not show

Fig. 2 Cultural morphology and microscopic picture of fungal endophyte Sh_26 grown on CYA and 2% MEA media (X = 800).
100
Fig. 3 Cultural morphology and microscopic picture of fungal endophyte Aa_27 grown on CYA and 2% MEA media (X = 800).
Fig. 4 Antimicrobial activities of fungal endophytes isolated
from Asclepias sinaica. C, control (without fungal inoculation);
Pc_25, Penicillium chrysogenum; Sh_26, Sterile hyphae; Aa_27,
Alternaria alternata.
any antimicrobial activity against the pathogenic microbes of
B. subtilis and E. coli.
Analysis of variance for the antimicrobial activity showed
that Pc_25 endophyte significantly inhibited the pathogenic
colonies of S. typhimurium and E. coli, compared to control
and Aa_27 endophytic treatments (P 6 0.001). Data also
showed that, there are no any inhibition zones have been
detected around the pathogenic microbial colonies of S. aureus,
B. subtilis, P. aeruginosa, and C. albicans due to the application
of fungal metabolites of endophyte isolate Pc_25.
A.H. Fouda et al.
Extracellular enzymatic activities
The recorded results revealed that Aa_27 endophyte had the
highest ability to produce all tested extracellular enzymes,
but this endophytic strain was recorded as negative tyrosinase
producer. The endophyte of Pc_25 exhibited extracellular
activity for amylase, pectinase, cellulase and xylanase;
whereas, the Sh_26 strain showed negative extracellular activ-
ity for amylase, xylanase, and gelatinase (Table 2).
Maximum amylase activity was recorded in Pc_25; how-
ever, there is no significant difference (P = 0.121) in amylase
activity was detected between Pc_25 and Aa_27. Sh_26 endo-
phytic strain did not show any amylase activity (Table 2).
The difference between pectinase activities of Pc_25 and
Sh_26 (P = 1.35) and between Pc_25 and Aa_27 (P = 1.354)
did not reach significant level. However pectinase activity of
Aa_27 was significantly higher as compared to those detected
by Sh_26 strain (P = 0.025).
Although maximum extracellular cellulase activity was
recorded in Pc_25 followed by Aa_27 and Sh_26 endophytic
strains, ANOVA analysis showed that variations between dif-
ferent endophytic strains to hydrolyze cellulose and CMC sub-
strates (P > 0.05) did not reach significant level.
Among the tested endophytes, Sh_26 strain showed nega-
tive xylanase activity, whereas Aa_27 significantly exhibited
high xylanolytic activity compared to Pc_25 (P 6 0.001).
Proteolytic activity of Aa_27 exerted the highest value,
compared to the other tested strains (P 6 0.001).
The production of extracellular tyrosinase was positive in
case of Aa_27 and Sh_26, and negative in case of Pc_25.
IAA production
Results presented in Fig. 6 showed that all the fungal endo-
phytes are able to produce IAA without tryptophan or by
Biotechnological applications of fungal endophytes 101

Fig. 5 Antimicrobial activities of fungal endophytes against some test organisms. C, control (without fungal inoculation); Pc_25,
Penicillium chrysogenum; Sh_26, Sterile hyphae; Aa_27, Alternaria alternata. Error bars are ±SE (n = 3). Different letters on bars denote
that mean values are significantly different (P 6 0.05) by Tukey LSD test (n = 3).

Table 2 Extracellular enzymatic activities of fungal endophytes using solid media.

Fungal isolate code Diameter of clear zones (mm) Tyrosinase
Amylase Pectinase Carboxy-methylcellulase Cellulase Xylanase Gelatinase
Control 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a ve
Pc_25 38.33 ± 1.66b 41.67 ± 1.66bc 41 ± 1b 38 ± 1.15b 25.33 ± 0.33b 0 ± 0a ve
Sh_26 0 ± 0a 40.67 ± 0.66b 40 ± 1.15b 38 ± 1b 0 ± 0a 0 ± 0a +ve
Aa_27 32 ± 3.03b 45.68 ± 0.67c 38.67 ± .66b 36.33 ± 1.2b 36.66 ± 0.65c 33.3 ± 1.65b +ve
In a column, values are the means ± SE followed by different letters are significantly different (P 6 0.05) by Tukey LSD test (n = 3).
102 A.H. Fouda et al.

Fig. 6 IAA production by fungal endophytes isolated from Asclepias sinaica at different concentrations of tryptophan. C, control
(without fungal inoculation); Pc_25, Penicillium chrysogenum; Sh_26, Sterile hyphae; Aa_27, Alternaria alternata. Error bars are ±SE
(n = 3). Different letters on bars denote that mean values are significantly different (P 6 0.05) by Tukey LSD test (n = 3).

using different concentrations of tryptophan as a precursor.

Accordingly, increase the tryptophan concentration in the Table 3 Root growth of maize plants and levels of ammonia
growth media resulted in the enhancement of IAA production, production as affected by inoculation with the three fungal
being maximum with 5 mg/ml of tryptophan (P 6 0.05). endophytic isolates.
Analysis of variance revealed that there was significant vari- Fungal isolate code Root length (cm) Ammonia production
ation in IAA production between the different endophytic
Control 9 ± 0.23a ve
inoculations. At growth media without tryptophan, the IAA
Pc_25 13.4 ± 0.24b High
production significantly differed between different fungal Sh_26 14.6 ± 0.25c Low
endophytes (F3,8 = 1422.9; P 6 0.001), where Pc_25 and Aa_27 14.4 ± 0.24c High
Sh_26 endophytes produced similar IAA contents
In a column, values are the means ± SE followed by different
(P = 0.19), which were significantly lower than those pro-
letters are significantly different (P 6 0.05) by Tukey LSD test
duced by Aa_27 (P 6 0.01). At 1 mg/ml tryptophan-growth (n = 5).
media, multi-comparison analysis (F3,8 = 423.9; P 6 0.001)
showed that Pc_25 and Aa_27 were similar IAA producers
(P = 0.996) and significantly higher than Sh_26 endophyte
(P 6 0.005) (Fig. 6). At growth media with tryptophan concen- Effect of fungal inoculation on root growth
trations of 2 mg/ml and 5 mg/ml, the analysis of variance was
(F3,8 = 203.6; P 6 0.001) and (F3,8 = 682.4; P 6 0.001), Analysis of variance showed that fungal endophytic inocula-
respectively. In these media, the maximum IAA production tion significantly affected root growth of maize plant
was recorded for Aa_27 followed by Pc_25, and then Sh_26 (F3,16 = 90.22; P 6 0.001) (Plate 2). The results revealed that
endophyte (P 6 0.05) (Fig. 6). inoculation with Sh_26 and Aa_27 had similar effect on root
The results showed also that all three fungal endophytes growth (P = 0.937), however both of these endophytes signif-
had the ability to produce ammonia in the growth media icantly increased root growth, compared to control and Pc_25
(Table 3). endophytic treatments (P 6 0.05). In addition, inoculation
Biotechnological applications of fungal endophytes
Plate 2 Effect of fungal endophytic inoculation on maize root
growth. C, control (without fungal inoculation); Pc_25,
Penicillium chrysogenum; Sh_26, Sterile hyphae; Aa_27,
Alternaria alternate.
with Pc_25 endophyte enhanced the root growth comparing
with control treatment (P 6 0.001) (Table 3).
Discussion
Three fungal endophytes were isolated from A. sinaica plant, a
medicinal native plant species in South Sinai- and identified as
P. chrysogenum Pc_25, A. alternata Aa_27, and unidentified
strain. Nonsporulating strain of fungal endophyte which failed
to sporulate was described as sterile hyphae Sh_26. This is the
common problem concerning with the identification of fungal
endophytes (Gamboa and Bayman, 2001; Promputtha et al.,
2005). These fungal endophytes have various biological and
biochemical properties potentially useful.
In the present study, the antimicrobial activity of the three
fungal endophytes isolated from A. sinaica plant revealed a sig-
nificant inhibitive effect against the selected bacterial and
fungal strains. The most antimicrobial activity was recorded
for Sh_26 endophyte which inhibits the growth of most coded
test organisms; however, Pc-25 and Aa_27 had inhibitory speci-
fic action against C. albicans and E. coli. Thereby, our investiga-
tions suggest that these fungal endophytes can be used as
producers of metabolites with broad and specific antimicrobial
activity. Kjer et al. (2009) isolated Alternaria sp. with antimicro-
bial activity from the mangrove plant, also Bertinetti et al.
(2009) recorded endophytic Penicillium sp. as antifungal
metabolite producer. Studies on endophytes for pharmaceutical
and biotechnological purposes are fundamental for the discov-
ery of new substances for human therapeutics including antibi-
otics, antimicotic, and anticarcinogenics (Bi et al., 2011).
Endophytes isolated from medicinal plant A. sinaica showed
bioactivity for broad spectrum of pathogenic microorganisms.
Similarly, Devaraju and Sathish (2011) assayed the bioactivity
of the endophytic microorganisms isolated from the medicinal
plant Mirabilis jalapa L.
103
The three fungal endophytes isolated in this study have
ability to produce various enzymes regarding to degrade
starch, gelatine, cellulose, pectin, xylan, and tyrosine. Aa_27
endophyte had high ability to produce most of the tested extra-
cellular enzymes, unlike to Sh_26 endophyte which only able
to degrade cellulose and pectin. Cellulolytic and pectinolytic
activities were prominent in all fungal endophytes where xyla-
nolytic activity was found in Pc_25, and Aa_27 endophytes;
similarly, Venkatesagowda et al. (2012) and Sunitha et al.
(2013) recorded high activity of cellulase and pectinase
enzymes in endophytic fungi isolated from oil-seeds and
medicinal plants. Production of hydrolytic extracellular
enzymes by fungal endophytes in particular cellulase and pecti-
nase acts as a bioactivity to obtain nutrients from hosts and
bio-resistance action against microbial pathogenic infection.
In the present study, Pc_25, and Aa_27 endophytes exhibited
maximum amylolytic activities, and Aa_27 was the only endo-
phyte isolated in our study was able to produce gelatinase
enzyme. Amirita et al. (2012) have been described 72% of
endophytes isolated from some medicinal plants as good amy-
lase and protease producers. Our finding supports that extra-
cellular enzymatic activity of endophytes plays potential role
to degrade polysaccharides and protein during the plant
senesce. In another view regarding to biotechnology, amy-
lolytic and proteolytic enzymes of endophytes are being inves-
tigated to improve industrial processes for polysaccharides and
protein biodegradation. The production of extracellular
enzymes by endophytes may imply a resistance strategy of
the host plant against pathogenic microorganisms, and in
another side to improve plant nutritional status (Choi et al.,
2005).
In the present study, fungal endophytes isolated from
A. sinaica plant possess different plant growth promoting
activities including ammonia and IAA production. Different
levels of IAA production were reported with various trypto-
phan concentrations. Although the obtained results also
revealed the ability of these fungal endophytes to produce
IAA in the absence of tryptophan i.e. the rate of IAA produc-
tion was related to the increase of tryptophan concentration.
Our findings of IAA production in the presence and absence
of tryptophan are in agreement with those of other researchers
(Ahmad et al., 2005). The three fungal endophytes showed dif-
ference in their capacity to produce ammonia and IAA; more-
over, inoculation with these fungal isolates significantly
enhanced the root growth. In addition, the effect of endophytic
inoculation on root growth significantly differed with different
endophytes, suggesting that the ability of these fungal endo-
phytes to enhance plant root growth may be mediated by their
effectiveness to produce ammonia and IAA. Zhang et al.
(2011) reported that different endophytes having different
ability for IAA production.
Conclusion
In the present investigation, three different fungal endophytes
were isolated from medicinal A. sinaica plant and identified as
P. chrysogenum Pc_25, A. alternata Aa_27 and Sterile hyphae
Sh_26. These fungal endophytes were found to have antimicro-
bial activity against various coded test organisms, and have
extracellular enzymatic activities to biodegrade different
polysaccharides and gelatine. In addition, these fungal strains
104
have the capacity to produce ammonia and IAA which poten-
tially influence the plant growth through various strategies.
For biotechnological and ecological applications of plant
growth promoting through fungal endophyte inoculation,
various molecular and biochemical studies are required to
investigate the role of endophytes to promote plant growth.
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